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The segregation of prokaryotic plasmids typically requires a centromere-like site and two proteins, a centromere-binding protein (CBP) and an NTPase. By contrast, a single 245 residue Par protein mediates partition of the prototypical staphylococcal multiresistance plasmid pSK1 in the absence of an identifiable NTPase component. To gain insight into centromere binding by pSK1 Par and its segregation function we performed structural, biochemical and in vivo studies. Here we show that pSK1 Par binds a centromere consisting of seven repeat elements. We demonstrate this Par-centromere interaction also mediates Par autoregulation. To elucidate the Par centromere binding mechanism, we obtained a structure of the Par N-terminal DNA-binding domain bound to centromere DNA to 2.25 Å. The pSK1 Par structure, which harbors a winged-helix-turn-helix (wHTH), is distinct from other plasmid CBP structures but shows homology to the B. subtilis chromosome segregation protein, RacA. Biochemical studies suggest the region C-terminal to the Par wHTH forms coiled coils and mediates oligomerization. Fluorescence microscopy analyses show that pSK1 Par enhances the separation of plasmids from clusters, driving effective segregation upon cell division. Combined the data provide insight into the molecular properties of a single protein partition system., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5'-GNNNNC-3' sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Horizontal transfer of plasmids encoding antimicrobial resistance and virulence determinants has been instrumental in Staphylococcus aureus evolution, including the emergence of community-associated methicillin-resistant S. aureus (CA-MRSA). In the early 1990s, the first CA-MRSA strain isolated in Western Australia (WA), WA-5, encoded cadmium, tetracycline, and penicillin resistance genes on plasmid pWBG753 (∼30 kb). WA-5 and pWBG753 appeared only briefly in WA; however, fusidic acid resistance plasmids related to pWBG753 were also present in the first European CA-MRSA isolates at the time. Here, we characterize a 72-kb conjugative plasmid, pWBG731, present in multiresistant WA-5-like clones from the same period. pWBG731 was a cointegrant formed from pWBG753 and a pWBG749 family conjugative plasmid. pWBG731 carried mupirocin, trimethoprim, cadmium, and penicillin resistance genes. The stepwise evolution of pWBG731 likely occurred through the combined actions of IS 257 , IS 257 -dependent miniature inverted-repeat transposable elements (MITEs), and the BinL resolution system of the β-lactamase transposon Tn 552 An evolutionarily intermediate ∼42-kb nonconjugative plasmid, pWBG715, possessed the same resistance genes as pWBG731 but retained an integrated copy of the small tetracycline resistance plasmid pT181. IS 257 likely facilitated the replacement of pT181 with conjugation genes on pWBG731, thus enabling autonomous transfer. Like conjugative plasmid pWBG749, pWBG731 also mobilized nonconjugative plasmids carrying oriT mimics. It seems likely that pWBG731 represents the product of multiple recombination events between the WA-5 pWBG753 plasmid and other mobile genetic elements present in indigenous community-associated methicillin-sensitive S. aureus (CA-MSSA) isolates. The molecular evolution of pWBG731 saliently illustrates how diverse mobile genetic elements can together facilitate rapid accrual and horizontal dissemination of multiresistance in S. aureus CA-MRSA., (Copyright © 2019 American Society for Microbiology.)

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation have been shaped in part by mobile genetic elements (MGEs), facilitating the global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs, in particular, how changes in the structure of multidrug resistance (MDR) plasmids may influence important staphylococcal phenotypes, is incomplete. Here, we undertook a population and functional genomics study of 212 methicillin-resistant S. aureus (MRSA) sequence type 239 (ST239) isolates collected over 32 years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have coevolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics, we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s and found that multiple structural variants have arisen. Plasmid maintenance and stability were linked to IS 256 - and IS 257 -mediated chromosomal integration and disruption of the plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin, trimethoprim, and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA isolates through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing the rates of gentamicin resistance and reduced chlorhexidine susceptibility and (ii) changes in the plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a potential evolutionary response of ST239 MRSA to biocides, one of which may contribute to the ongoing persistence and adaptation of this lineage within health care institutions., (Copyright © 2019 Baines et al.)

Strains of Staphylococcus aureus , and to a lesser extent other staphylococcal species, are a significant cause of morbidity and mortality. An important factor in the notoriety of these organisms stems from their frequent resistance to many antimicrobial agents used for chemotherapy. This review catalogues the variety of mobile genetic elements that have been identified in staphylococci, with a primary focus on those associated with the recruitment and spread of antimicrobial resistance genes. These include plasmids, transposable elements such as insertion sequences and transposons, and integrative elements including ICE and SCC elements. In concert, these diverse entities facilitate the intra- and inter-cellular gene mobility that enables horizontal genetic exchange, and have also been found to play additional roles in modulating gene expression and genome rearrangement.

Conjugation is a dominant mechanism of horizontal gene transfer and substantially contributes to the plasticity and evolvability of prokaryotic genomes. The impact of conjugation on genetic flux extends well beyond self-transmissible conjugative elements, because non-conjugative 'mobilizable elements' utilize other elements' conjugative apparatus for transfer. Bacterial genome comparisons highlight plasmids as vehicles for dissemination of pathogenesis and antimicrobial-resistance determinants, but for most non-conjugative plasmids, a mobilization mechanism is not apparent. Recently we discovered many Staphylococcus aureus plasmids lacking mobilization genes carry oriT sequences that mimic those on conjugative plasmids, suggesting that significantly more elements may be mobilizable than previously recognized. Here we summarize our findings, review the diverse mobilization strategies employed by mobile genetic elements and discuss implications for future gene-transfer research., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Actin-like proteins (Alps) are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

Unlabelled: Antimicrobial resistance in Staphylococcus aureus presents an increasing threat to human health. This resistance is often encoded on mobile plasmids, such as pSK41; however, the mechanism of transfer of these plasmids is not well understood. In this study, we first examine key protein-DNA interactions formed by the relaxase enzyme, NES, which initiates and terminates the transfer of the multidrug resistance plasmid pSK41. Two loops on the NES protein, hairpin loops 1 and 2, form extensive contacts with the DNA hairpin formed at the oriT region of pSK41, and here we establish that these contacts are essential for proper DNA cleavage and religation by the full 665-residue NES protein in vitro. Second, pSK156 and pCA347 are nonconjugative Staphylococcus aureus plasmids that contain sequences similar to the oriT region of pSK41 but differ in the sequence predicted to form a DNA hairpin. We show that pSK41-encoded NES is able to bind, cleave, and religate the oriT sequences of these nonconjugative plasmids in vitro. Although pSK41 could mobilize a coresident plasmid harboring its cognate oriT, it was unable to mobilize plasmids containing the pSK156 and pCA347 variant oriT mimics, suggesting that an accessory protein like that previously shown to confer specificity in the pWBG749 system may also be involved in transmission of plasmids containing a pSK41-like oriT. These data indicate that the conjugative relaxase in trans mechanism recently described for the pWBG749 family of plasmids also applies to the pSK41 family of plasmids, further heightening the potential significance of this mechanism in the horizontal transfer of staphylococcal plasmids., Importance: Understanding the mechanism of antimicrobial resistance transfer in bacteria such as Staphylococcus aureus is an important step toward potentially slowing the spread of antimicrobial-resistant infections. This work establishes protein-DNA interactions essential for the transfer of the Staphylococcus aureus multiresistance plasmid pSK41 by its relaxase, NES. This enzyme also processed variant oriT-like sequences found on numerous plasmids previously considered nontransmissible, suggesting that in conjunction with an uncharacterized accessory protein, these plasmids may be transferred horizontally via a relaxase in trans mechanism. These findings have important implications for our understanding of staphylococcal resistance plasmid evolution., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

pSK41 is a prototypical 46-kb conjugative multiresistance plasmid of Staphylococcus aureus. The pSK41 replication initiation protein (Rep) is rate-limiting for plasmid replication, and its expression is negatively regulated by a small, non-coding antisense transcript, RNAI, that is complementary to the rep mRNA leader region. In this study, enzymatic probing was used to verify the predicted secondary structures of RNAI and its target RNA. We demonstrated that two stem-loop structures of RNAI, SLRNAI-II and SLRNAI-III, were important for inhibition. A putative U-turn motif detected in the loop of SLrep-I (5'-UUGG-3') was analysed for its significance to RNAI-mediated inhibition in vivo and Northern blotting suggested that rep mRNA was processed. Taken together, these observations support our previously proposed model but also raise new questions about the replication control mechanism., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The staphylococcal multiresistance plasmids are key contributors to the alarming rise in bacterial multidrug resistance. A conserved replication initiator, RepA, encoded on these plasmids is essential for their propagation. RepA proteins consist of flexibly linked N-terminal (NTD) and C-terminal (CTD) domains. Despite their essential role in replication, the molecular basis for RepA function is unknown. Here we describe a complete structural and functional dissection of RepA proteins. Unexpectedly, both the RepA NTD and CTD show similarity to the corresponding domains of the bacterial primosome protein, DnaD. Although the RepA and DnaD NTD both contain winged helix-turn-helices, the DnaD NTD self-assembles into large scaffolds whereas the tetrameric RepA NTD binds DNA iterons using a newly described DNA binding mode. Strikingly, structural and atomic force microscopy data reveal that the NTD tetramer mediates DNA bridging, suggesting a molecular mechanism for origin handcuffing. Finally, data show that the RepA CTD interacts with the host DnaG primase, which binds the replicative helicase. Thus, these combined data reveal the molecular mechanism by which RepA mediates the specific replicon assembly of staphylococcal multiresistant plasmids.

Plasmid pSK41 is a large, low-copy-number, conjugative plasmid from Staphylococcus aureus that is representative of a family of staphylococcal plasmids that confer multiple resistances to a wide range of antimicrobial agents. The plasmid consists of a conserved plasmid backbone containing the genes for plasmid housekeeping functions, which is punctuated by copies of IS257 that flank a Tn4001-hybrid structure and cointegrated plasmids that harbour resistance genes. This review summarises the current understanding of the biology of pSK41, focussing on the systems responsible for its replication, maintenance and transmission, and their regulation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

Replication of staphylococcal multiresistance plasmid pSK41 is initiated by binding of the replication initiator protein (Rep) to the Rep boxes, a series of four direct repeats located centrally within the rep gene. A Staphylococcus aureus strain was engineered to provide Rep in trans, allowing localization of the pSK41 origin of replication (oriV) to a 185 bp segment, which included the Rep boxes and a series of downstream direct repeats. Deletion analysis of individual Rep boxes revealed that all four Rep boxes are required for maximum origin activity, with the deletion of one or more Rep boxes having a significant effect on the proficiency of replication. However, a hierarchy of importance was identified among the Rep boxes, which appears to be mediated by the minor sequence variations that exist between them. DNA binding studies with truncated Rep proteins have enabled the DNA binding domain to be localized to the N-terminal 134 amino acids of the protein.

We have established a plasmid-based system that enables tightly controlled gene expression and the generation of GFP fusion proteins in Staphylococcus aureus simply and rapidly. This system takes advantage of an Escherichia coli-S. aureus shuttle vector that contains the replication region of the S. aureus theta-mode multiresistance plasmid pSK41, and is therefore a stable low-copy-number plasmid in the latter organism. This vector also contains a multiple cloning site downstream of the IPTG-inducible Pspac promoter for insertion of the gene of interest. Production of encoded proteins can be stringently regulated in an IPTG-dependent manner by introducing a pE194-based plasmid, pGL485, carrying a constitutively expressed lacI gene. Using GFP fusions to two essential proteins of S. aureus, FtsZ and NusA, we showed that our plasmid allowed tightly controlled gene expression and accurate localization of fusion proteins with no detrimental effect on cells at low inducer concentrations. At higher IPTG concentrations, we obtained sixfold overproduction of protein compared with wild-type levels, with FtsZ-GFP-expressing cells showing lysis and delocalized fluorescence, while NusA-GFP showed only delocalized fluorescence. These results show that our system is capable of titratable induction of gene expression for localization or overexpression studies.

We have sequenced the conjugative plasmid pPR9, which carries the ileS2 gene, which had contributed to the dissemination of high-level mupirocin resistance at our institution. The plasmid backbone shows extensive genetic conservation with plasmids belonging to the pSK41/pGO1 family, but comparative analyses have revealed key differences that provide important insights into the evolution of these medically important plasmids and high-level mupirocin resistance in staphylococci and highlight the role of insertion sequence IS257 in these processes.

Type II plasmid partition systems utilize ParM NTPases in coordination with a centromere-binding protein called ParR to mediate accurate DNA segregation, a process critical for plasmid retention. The Staphylococcus aureus pSK41 plasmid is a medically important plasmid that confers resistance to multiple antibiotics, disinfectants, and antiseptics. In the first step of partition, the pSK41 ParR binds its DNA centromere to form a superhelical partition complex that recruits ParM, which then mediates plasmid separation. pSK41 ParM is homologous to R1 ParM, a known actin homologue, suggesting that it may also form filaments to drive partition. To gain insight into the partition function of ParM, we examined its ability to form filaments and determined the crystal structure of apoParM to 1.95 A. The structure shows that pSK41 ParM belongs to the actin/Hsp70 superfamily. Unexpectedly, however, pSK41 ParM shows the strongest structural homology to the archaeal actin-like protein Thermoplasma acidophilum Ta0583, rather than its functional homologue, R1 ParM. Consistent with this divergence, we find that regions shown to be involved in R1 ParM filament formation are not important in formation of pSK41 ParM polymers. These data are also consonant with our finding that pSK41 ParM forms 1-start 10/4 helices very different from the 37/17 symmetry of R1 ParM. The polymerization kinetics of pSK41 ParM also differed from that of R1 ParM. These results indicate that type II NTPases utilize different polymeric structures to drive plasmid segregation.

The pheromone-responsive conjugative plasmids of Enterococcus faecalis and the multiresistance plasmids pSK1 and pSK41 of Staphylococcus aureus are among the best studied plasmids native to Gram-positive bacteria. Although these plasmids seem largely restricted to their native hosts, protein sequence comparison of their replication initiator proteins indicates that they are clearly related. Homology searches indicate that these replicons are representatives of a large family of plasmids and a few phage that are widespread among the low G+C Gram-positive bacteria. We propose to name this family the RepA_N family of replicons after the annotated conserved domain that the initiator protein contains. Detailed sequence comparisons indicate that the initiator protein phylogeny is largely congruent with that of the host, suggesting that the replicons have evolved along with their current hosts and that intergeneric transfer has been rare. However, related proteins were identified on chromosomal regions bearing characteristics indicative of ICE elements, and the phylogeny of these proteins displayed evidence of more frequent intergeneric transfer. Comparison of stability determinants associated with the RepA_N replicons suggests that they have a modular evolution as has been observed in other plasmid families.

Multidrug-resistant staphylococci often harbour plasmids that carry genes conferring resistance to several antimicrobial compounds. Many of these multiresistance plasmids appear to utilize a related theta-type replication system for which multiresistance plasmid pSK1 serves as a prototype. Essential pSK1 replication elements were identified by cloning segments of the replication region and testing the resulting plasmids for replication proficiency. An iterated region within rep and a DNA segment of up to 68 bp upstream of the rep promoter were both found to be essential for origin activity. The Rep protein was overexpressed as a 6xHis-tagged C-terminal fusion protein and was shown to bind in vitro to four Rep boxes located within the rep coding region. Inactivation of a divergently oriented promoter upstream of rep, designated P(rnaI), resulted in an elevated plasmid copy number. Comparative analyses suggest that the replication systems of many staphylococcal multiresistance plasmids share a similar genetic organization and utilize an antisense-RNA-mediated regulatory mechanism for copy number control.

The stable inheritance of genetic material depends on accurate DNA partition. Plasmids serve as tractable model systems to study DNA segregation because they require only a DNA centromere, a centromere-binding protein and a force-generating ATPase. The centromeres of partition (par) systems typically consist of a tandem arrangement of direct repeats. The best-characterized par system contains a centromere-binding protein called ParR and an ATPase called ParM. In the first step of segregation, multiple ParR proteins interact with the centromere repeats to form a large nucleoprotein complex of unknown structure called the segrosome, which binds ParM filaments. pSK41 ParR binds a centromere consisting of multiple 20-base-pair (bp) tandem repeats to mediate both transcription autoregulation and segregation. Here we report the structure of the pSK41 segrosome revealed in the crystal structure of a ParR-DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously to generate the full-length centromere with the ribbon-helix-helix (RHH) fold of ParR binding successive DNA repeats as dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous protein super-helical array, wrapping the DNA about its positive convex surface to form a large segrosome with an open, solenoid-shaped structure, suggesting a mechanism for ParM capture and subsequent plasmid segregation.

Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (PrnaI) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when PrnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.

The vast majority of large staphylococcal plasmids characterized to date appear to possess an evolutionarily common replication system, which has clearly had a major impact on the evolution of antimicrobial resistant staphylococci worldwide. Related systems have also been found in plasmids from other Gram-positive genera, including enterococci, streptococci and bacilli. The 46.4 kb plasmid pSK41 is the prototype of a family of conjugative staphylococcal multiresistance plasmids. The replication region of pSK41 encodes a protein product, Rep, which was shown to be essential for replication; mutations that truncated Rep could be complemented in trans. Rep was found to bind in vitro to four tandem repeat sequences located centrally within the rep coding region. An A + T-rich inverted repeat sequence upstream of rep was required for efficient replication, whereas no sequences downstream of rep were necessary. An antisense countertranscript, RNAI, encoded upstream of rep was identified and transcriptional start points for both RNAI and the rep-mRNA were defined.

The orf245 gene is located immediately upstream of, and divergently transcribed from, the replication initiation gene, rep, of the Staphylococcus aureus multiresistance plasmid pSK1, and related genes have been found in association with a range of evolutionarily distinct replication genes on plasmids from various gram-positive genera. orf245 has been shown previously to extend the segregational stability of a pSK1 minireplicon. Here we describe an investigation into the basis of orf245-mediated stabilization. orf245 was not found to influence transcription of pSK1 rep, indicating that it is not directly involved in plasmid replication. This was confirmed by demonstrating that orf245 is able to enhance the segregational stability of heterologous theta- and rolling-circle-replicating replicons, suggesting that it encodes a plasmid maintenance function. Evidence inconsistent with postsegregational killing and multimer resolution mechanisms was obtained; however, the intergenic region upstream of orf245 was found to mediate orf245-dependent incompatibility, as would be expected if it encodes a cis-acting centromere-like site. Taken together, these findings implicate active partitioning as the probable basis of the activity of orf245, which is therefore redesignated par. Since it is unrelated to any gene known to play a role in plasmid segregation, it seems likely that pSK1 par potentially represents the prototype of a novel class of active partitioning systems that are distinguished by their capacity to enhance plasmid segregational stability via a single protein-encoding gene.

A series of Staphylococcus aureus-Escherichia coli shuttle vectors were constructed which contained the replication and maintenance functions of the S. aureus theta-mode multiresistance plasmid pSK1. The utility of the newly constructed vectors was demonstrated by the successful cloning and expression of several genes that had previously proven difficult to express in S. aureus. Additional vectors which permit the generation of transcriptional and translational fusions to an S. aureus blaZ reporter gene were also produced and subsequently employed to determine the relative strengths in S. aureus of a number of promoters. By utilizing the theta-mode replication functions of pSK1, the shuttle vectors described largely avoided the segregational and structural stability problems frequently encountered with Gram-positive rolling-circle-based vectors. In addition, these plasmids represent vectors which are suitable for the analysis of genes in S. aureus at low copy number.

pED208 is a transfer-derepressed mutant of the IncFV plasmid, F(0)lac, which has an IS2 element inserted in its traY gene, resulting in constitutive overexpression of its transfer (tra) region. The pED208 transfer region, which encodes proteins responsible for pilus synthesis and conjugative plasmid transfer, was sequenced and found to be very similar to the F tra region in terms of its organization although most pED208 tra proteins share only about 45% amino acid identity. All the essential genes for F transfer had homologs within the pED208 transfer region with the exception of traQ, which encodes the chaperone for stable F-pilin expression. F(0)lac appears to have a fertility inhibition system different than the FinOP system of other F-like plasmids, and its transfer efficiency was increased in the presence of F or R100, suggesting that it could be mobilized by these plasmids. The F-like transfer systems specified by F, R100, and F(0)lac were highly specific for their cognate origins of transfer (oriT) as measured by their abilities to mobilize chimeric oriT-containing plasmids.

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.

Analyses of the Staphylococcus epidermidis multiresistance plasmids pSK697 and pSK818 have revealed them to be closely related to the trimethoprim resistance plasmid pSK639, also isolated from S. epidermidis. pSK697 and pSK818 were found to contain a cointegrated copy of a second plasmid related to the S. epidermidis multidrug antiseptic and disinfectant resistance plasmid pSK108 and the S. aureus tetracycline resistance plasmid pT181, respectively. In contrast to pSK639, both plasmids were found to contain a third copy of IS257, such that the integrated plasmids in both cases are flanked by a copy of this element. This organization and the presence of duplicated sequences at the extremities of the integrated plasmids implicate IS257 in the formation of these cointegrate plasmids. Sequence analysis of the IS257 elements from these plasmids has provided insights into the probable mechanism of cointegration, viz., nonresolved replicative transposition of IS257.

The 2.4-kb Staphylococcus epidermidis plasmid, pSK108, encodes a qacC multidrug resistance determinant. Sequence analysis has revealed that pSK108 is a member of the pC194 family of rolling circle replicating plasmids and suggests that the DNA segment containing qacC, which is bounded by the replication nick site and the minus origin palA, represents a resistance gene cassette that has undergone horizontal genetic exchange.

The transposon-like structure Tn4003 and related elements were found to encode high- and low-level trimethoprim resistance (Tpr) in Staphylococcus aureus and coagulase-negative staphylococci. By using transcriptional fusions in Escherichia coli, the variation in resistance levels was found to correlate with the transcriptional activity of the region presumed to carry the promoter for the operon containing the Tpr dihydrofolate reductase gene, dfrA, encoded by these elements. The reduced transcriptional activities exhibited by elements encoding low-level Tpr appear to be a consequence of deletions adjacent to the copy of IS257 which normally encodes the -35 sequences of these promoters. The data obtained not only support the involvement of IS257 in the transcription of the proposed thyE-dfrA-orf-140 operon of Tn4003 but may also implicate this insertion sequence in the mechanisms resulting in the variation in Tpr levels observed in staphylococci.

The traH gene of the staphylococcal conjugative plasmid pSK41 has recently been shown to encode a lipoprotein (N. Firth, K. P. Ridgway, M. E. Byrne, P. D. Fink, L. Johnson, I. T. Paulsen, and R. A. Skurray, Gene 136:13-25, 1993). Here we report that traH encodes a product recognized as a pheromone by Enterococcus faecalis cells harboring the conjugative plasmid pAD1. The mature traH product is not essential for this phenomenon, as expression of pheromone-like activity was found to require sequences encoding only the pro-TraH signal peptide.

The nucleotide sequence of a 14.4-kb region (tra) associated with DNA transfer of the staphylococcal conjugative plasmid, pSK41, has been determined. Analysis of the sequence revealed the presence of 15 genes potentially involved in the conjugative process. Polypeptide products likely to correspond to ten of these genes have been identified, of which one was found to be a lipoprotein. Comparison of the deduced tra products to the protein databases revealed several interesting similarities, one of which suggests an evolutionary link between this Gram+ bacterial conjugation system and DNA transfer systems of Gram- bacteria, such as Escherichia coli and Agrobacterium tumefaciens. The nt sequence also provided an insight into the transcriptional organisation and regulation of the region.

The nucleotide sequence of the F plasmid transfer gene traH, which is involved in F-pilus assembly in Escherichia coli K-12, has been determined. From the sequence data, it would appear that traH encodes a 38,897-dalton precursor polypeptide which is processed to give a periplasmic protein. Furthermore, a new gene, trbF, has been located immediately upstream of traH and shown to be expressed by means of a translational fusion to lacZ. Using galK fusion and S1 nuclease protection studies, a weak traJ-dependent promoter, P trbF, has been mapped upstream and adjacent to trbF. Transcription of trbF and traH from P trbF may well serve to complement transcription from the major tra operon promoter PY located some 16 kb upstream of these genes.